NADH-methemoglobin Reductase Research Articles

Favism Induced Methemoglobinemia in G6DP Deficient Patients: Case Series and Review of Literature

Introduction: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a well-known cause of hemolysis. It has a notable prevalence in African, Asian, and Mediterranean countries. Favism is a common trigger of oxidative stress in G6PD deficient people, which can lead to hemolysis. Additionally, fava bean ingestion can cause methemoglobinemia (MethHgb), an abnormal variation in the Hgb in which the ferrous (Fe2+) iron in heme is oxidized to the ferric (Fe3+) state. It is rare to have both G6PD deficiency and MethHgb secondary to favism at the same time. The first-line treatment for MethHgb is methylene blue. However, in G6PD deficient patients, it can potentiate hemolysis. Methods: We reviewed the literature using PubMed and Google scholar and found 6 cases of MethHgb secondary to favism in patients with G6PD deficiency. We also analyzed two cases which are still unpublished, making a total of 8 cases. Results: All 8 cases were male with median age of 18 years (1 - 56). 1 patient had a prior diagnosis of G6PD deficiency while 7 were newly diagnosed. Median Hgb was 8.3gm/dL (4.6 - 12.5) and median MetHgb was 7.8 % (3.5 - 35). 1 patient received methylene blue, and 4 received Vitamin C. All of the patients recovered and were discharged [Table 1]. Unpublished case 1: A 56-year-old male presented with MethHgb and hemolytic anemia, secondary to fava bean ingestion. Hgb on admission and lowest recorded were 9.9 and 6.5 gm/dL, respectively. He had an SPO2 of 70% on room air and 101.2 % on ABG. Methylene blue administration worsened the hemolysis as he was G6PD deficient but not diagnosed before. He got better with discontinuation of methylene blue and Vitamin C and was discharged on day 5. Unpublished case 2: A 43-year-old male, known case of G6PD deficiency presented with MethHgb and hemolytic anemia, secondary to fava bean ingestion. Hgb on admission and lowest recorded were 12.5 and 7.4 gm/dL, respectively. He had an SPO2 of 82% on room air and 100 % on ABG. He received IV vitamin C and recovered and was discharged on day 4. Discussion: Methemoglobinemia is usually acquired, secondary to oxidative stress in the body, but can rarely be congenital. Enzyme systems such as NADH methemoglobin reductase, NADPH methemoglobin reductase, ascorbic acid, and glutathione reductase systems keep a check on the accumulation of methemoglobin in the blood. However, these mechanisms can be insufficient to counter the conversion of Hgb to MethHgb, consequently promoting an oxidative state in the body. It can be due to the overproduction of methemoglobin (secondary to exposure to certain drugs, chemicals, or food items, but can sometimes be hereditary) or under conversion to Hgb due to unavailable enzyme mechanisms. One of the causes of the inability to counteract methemoglobin can be secondary to G6PD deficiency. Patients with MetHgb have a low oxygen saturation (SPO2) on pulse oximeters but a falsely high SPO2 on arterial blood gasses (ABG). The treatment depends on symptoms and the level of MethHgb. The first step is to remove any possible precipitator if present. Symptomatic patients (and asymptomatic with a level of methemoglobin >30 %) are treated with methylene blue (1-2mg/kg), which is reduced to leuko-methylene blue via NADPH dependent methemoglobin reductase. This, in turn, reduces methemoglobin back to Hgb, correcting the abnormality [Figure 1]. Rarely, patients can present with co-occurrence of MethHgb and G6PD deficiency. In such cases, caution is required while giving methylene blue as they do not have sufficient levels of NADPH to reduce it. Otherwise, a cascade of oxidative hemolysis ensues secondary to underlying G6PD deficiency, resulting in a vicious cycle of further methemoglobinemia. The most frequent cause of this co-occurrence is the ingestion of fava beans, which can simultaneously induce MethHgb and potentiate G6PD deficiency. One of our patients had a history of favism without developing any symptoms. Only this time, he ate fava beans in a larger amount, leading to hemolysis and MethHgb. Conclusion: Favism is a rare cause of the co-occurrence of methemoglobinemia and hemolysis in G6PD deficient individuals. It is vital to identify G6PD deficiency in patients presenting with MethHgb, as the initiation of methylene blue in such individuals can result in a cascade of oxidative hemolysis. A history of fava beans ingestion without any symptoms does not rule out G6PD deficiency, as it is proportional to the number of beans ingested. Disclosures No relevant conflicts of interest to declare.

A CASE OF DAPSONE-INDUCED METHEMOGLOBINEMIA

SESSION TITLE: Wednesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/23/2019 09:45 AM - 10:45 AM INTRODUCTION: Dapsone is an antibiotic used with relative frequency for dermatological illnesses and opportunistic infection prophylaxis. A known complication of this drug is methemoglobinemia, a condition that can be both difficult to identify and potentially fatal. Here we present the case of a patient diagnosed with and treated for dapsone-induced methemoglobinemia. CASE PRESENTATION: A 40-year-old Caucasian female with a history of depression complicated by past suicide attempt and chronic autoimmune urticaria treated with dapsone presented to the emergency department with suicidal ideation. She reported taking 45 pills of 100mg dapsone and 27 pills of 300mg gabapentin prior to arrival. Physical exam was remarkable for cyanosis noted in the perioral area. Her initial course was complicated by oxygen desaturation to 83% with no identified pulmonary process. She was found to have methemoglobinemia with initial methemoglobin level of 39.9% on VBG. She was treated with 50mg IV methylene blue in the Emergency Department then transferred to the Intensive Care Unit. Throughout her admission, she was given a total of 300mg IV methylene blue with eventual resolution of methemoglobinemia and symptoms. DISCUSSION: Acquired methemoglobinemia is thought to be due exposure to oxidizing agents such as dapsone and other commonly used medications. These agents accelerate the oxidization of hemoglobin at a rate that overwhelms endogenous reduction capacities. This results in an inability of hemoglobin to bind oxygen, ultimately leading to reduced oxygen delivery throughout the body and diffuse tissue hypoxia. Methylene blue, a common antidote to methemoglobinemia, works within the NADH-Methemoglobin reductase system to reduce methemoglobin to hemoglobin. Clinically, cyanosis resistant to oxygen therapy will typically occur at methemoglobin levels of 15% or higher. As methemoglobin levels increase, symptoms such as headache, fatigue and tachycardia become more common. Arrhythmias, seizures and coma typically occur at levels above 60%, with death possible over 70%. Diagnosis of methemoglobinemia can be somewhat challenging and requires a degree of clinical suspicion. Standard pulse oximetry does not detect methemoglobin and can reflect a falsely elevated or falsely decreased oxygen saturation depending on concentration. Co-oximetry can help with diagnosis but is not as definitive as arterial blood gas in the hospital setting. Patients diagnosed with methemoglobinemia should be treated with high-dose oxygen as well as methylene blue for fractions greater than 30% or symptomatic hypoxia. For patients with G6PD deficiency, however, methylene blue is contraindicated. Hyperbaric oxygen and exchange transfusions are safe alternatives and useful for severe or refractory cases. CONCLUSIONS: Methemoglobinemia is a potentially life-threatening condition. Clinicians should be aware of the signs, symptoms and treatment options. Reference #1: Pereira, R.O. and Costa, J., 2008. Methemoglobinemia: from diagnosis to treatment. Revista brasileira de anestesiologia, 58(6), pp.651-664. Reference #2: Longo, D. and Fauci, A., 2013. Poisoning and drug overdose. Harrison's manual of medicine. (pp. 177). McGraw Hill Professional. Reference #3: Marino, P. (2014). Oximetry and capnometry. The ICU Book (pp. 409-413). Philadelphia, PA: Wolters Kluwer Health/Lippincott Williams & Wilkins. DISCLOSURES: No relevant relationships by Abigail Go, source=Web Response No relevant relationships by Stephen Osmon, source=Web Response No relevant relationships by Maria Srour, source=Web Response

Bioinformatic identification of cytochrome b5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b5 in parasitic adaptation

We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respectively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b5, an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b5 and the canonical cytochrome b5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cytochrome b5-like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b5, which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b5 in parasitic adaptation.

아질산에 노출된 뱀장어(Anguilla japonica)의 혈액학적 반응과 항산화효소의 활성 변화

The study was performed to investigate changes in hematological responses and antioxidative enzyme activities (superoxide dismutase, SOD; catalase, CAT) of Japanese eel Anguilla japonica following exposure to 0 (control), 2.33, 4.60, 6,64 and 8.78 mM nitrite-N in fresh water for 48 h. Hematological parameters such as plasma nitrite, electrolytes, cortisol, glucose, glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT), hemoglobin (Hb), methemoglobin (metHb) and NADH-methemoglobin reductase (NMR) were measured. Plasma nitrite, cortisol, metHb and NMR increased directly with increasing ambient nitrite concentration, while Hb content showed a progressive decline. Levels of plasma potassium, GOT and GPT of the eel exposed to 6.64 mM ambient nitrite were significantly higher than the control fish. The activity of SOD and CAT in plasma, gill and liver of the eel following exposure to nitrite were augmented by increasing ambient nitrite. Levels of plasma nitrite, metHb, NMR, cortisol, glucose and antioxidative enzyme activities of the eel exposed to 2.33 mM ambient nitrite were significantly higher than the control fish. This study suggested that the eel acutely exposed to elevated ambient nitrite causes nitrite-induced stress responses, changes in antioxidative enzyme activities and hematological parameters.

The effects of chronic nitrate supplementation on erythrocytic methaemoglobin reduction in cattle

Calcium nitrate and urea were fed as a supplement on an isonitrogenous basis to Angus steers and their erythrocytic methaemoglobin concentrations and NADH- and NADPH-methaemoglobin reductase levels were measured over a 54-day period. Methaemoglobin concentrations remained elevated despite increases in NADH-methaemoglobin reductase activity. In a second experiment, Brahman cross steers were fed either calcium nitrate or urea supplements for 111 days. Blood cells were then taken, washed and exposed to sodium nitrite to convert all haemoglobin to methaemoglobin. The rates of glycolysis and methaemoglobin reduction were measured following incubation of these cells in buffers containing 1, 5 or 10 mM inorganic phosphate. Glucose consumption and methaemoglobin reduction were increased by inorganic phosphate and were more rapid in those animals supplemented with nitrate. Lactate production of erythrocytes was reduced in those animals fed nitrate. It is concluded that adaptation to chronic nitrite exposure occurs in the erythron, resulting in greater methaemoglobin reduction potential and that there is competition between NADH-methaemoglobin reductase and lactate dehydrogenase for NADH.

Activities of three erythrocyte enzymes of hyperglycemic rats (Rattus norvegicus) treated with Allium sativa extract.

BackgroundThe present study sought to investigate erythrocyte glutathione S-transferases (GST), NADH-Methaemoglobin reductase (NADH-MR) and Na+/K+-ATPase activities of hypoglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa as agent of glycemic control.MethodsHyperglycemia was induced by a single intra-peritoneal injection of 0.1 mol/L alloxan monohydrate in phosphate buffer saline (PBS) solution (pH = 7.4); dosage = 140 mg/kg. At the end of the experimental time (t = 76 h), erythrocyte GST, NADH-MR and Na+/K+-ATPase activities as well as serum fasting blood sugar (FBS) levels were measured by spectrophotometric methods.ResultsSerum FBS levels of control/normal (C/N) rats ranged between 72.93 ± 0.82–95.12 ± 0.92 mg/dL, whereas experimental rats without glycemic control gave: 249.41 ± 1.03–256.11 ± 1.23 mg/dL. Hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa exhibited comparative reduced serum levels of FBS alongside with erythrocyte GST, NADH-MR and Na+/K+-ATPase activities. The average relative activities of the three enzymes and corresponding order of enzyme activity in hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa was: NADH-MR = 60.99% > GST = 47.81% > Na+/K+-ATPase = 46.81%. In the same order, relative activities of the three enzymes in rats without glycemic control were: NADH-MR = 49.65% > GST = 23.69% > Na+/K+-ATPase = 17.02%.ConclusionErythrocyte GST, NADH-MR and Na+/K+-ATPase activities gave insights into the pathophysiology of diabetic state and served as biomarkers for ascertaining therapeutic control in Type 1 diabetes mellitus.

Activities of Three Erythrocyte Enzymes of Hyperglycemic Rats (Rattus norvegicus) Treated with Allium sativa Extract.

The present study sought to investigate erythrocyte glutathione S-transferases (GST), NADH-Methaemoglobin reductase (NADH-MR) and Na+/K+-ATPase activities of hypoglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa as agent of glycemic control. Hyperglycemia was induced by a single intra-peritoneal injection of 0.1 mol/L alloxan monohydrate in phosphate buffer saline (PBS) solution (pH = 7.4); dosage = 140 mg/kg. At the end of the experimental time (t = 76 h), erythrocyte GST, NADH-MR and Na+/K+-ATPase activities as well as serum fasting blood sugar (FBS) levels were measured by spectrophotometric methods. Serum FBS levels of control/normal (C/N) rats ranged between 72.93 ± 0.82–95.12 ± 0.92 mg/dL, whereas experimental rats without glycemic control gave: 249.41 ± 1.03–256.11 ± 1.23 mg/dL. Hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa exhibited comparative reduced serum levels of FBS alongside with erythrocyte GST, NADH-MR and Na+/K+-ATPase activities. The average relative activities of the three enzymes and corresponding order of enzyme activity in hyperglycemic rats treated with ethanol/water (1:2 v/v) extract of A. sativa was: NADH-MR = 60.99% > GST = 47.81% > Na+/K+-ATPase = 46.81%. In the same order, relative activities of the three enzymes in rats without glycemic control were: NADH-MR = 49.65% > GST = 23.69% > Na+/K+-ATPase = 17.02%. Erythrocyte GST, NADH-MR and Na+/K+-ATPase activities gave insights into the pathophysiology of diabetic state and served as biomarkers for ascertaining therapeutic control in Type 1 diabetes mellitus.

Methemoglobinemia: What the anaesthetist must know

Sir, Methemoglobinemia is an uncommon but potentially serious haemoglobin disorder in which oxygenation product of haemoglobin, i.e., methemoglobin accumulates in erythrocytes in large amounts. It is not capable of carrying oxygen and thus interferes with oxygen delivery to the tissues.[12] A 1½-year-old child was posted for cystoscopy and dilatation for urethral stricture. He was referred to our centre with complaints of difficulty in micturition and bluish discoloration of nails and lips. Cardiorespiratory examination revealed no abnormality. Saturation measured 83% on room air. Further cardiopulmonary work-up, including echocardiography, pulmonary angiography were normal. Haemoglobin electrophoresis revealed absence of abnormal form of haemoglobin and glucose-6-phosphate dehydrogenase (G6PD) level was normal. Methemoglobin level in the blood was 5% (normal value being < 1%). Arterial blood gas (ABG) analysis on room air showed pH: 7.3, pCO2: 25, pO2: 56, HCO3: 14, BE: −8.3 and SaO2: 87.8%. Perioperatively ascorbic acid 60 mg tds was started. Child was premedicated with glycopyrrolate 30 mcg intravenous (IV), midazolam 0.3 mg IV and ketamine 4 mg IV. Methylene blue ampoule was kept ready in operation theatre. Anaesthesia was induced with fentanyl 15 mcg, propofol 15 mg and atracurium 4 mg. No 1.5 proseal laryngeal mask airway was inserted after lubricating with liquid paraffin. Liquid paraffin was also used for lubrication of surgical instruments and cystoscope. Anaesthesia was maintained with 100% oxygen, isoflurane and controlled ventilation. Standard non-invasive monitors were used. Co-oximetry was not available. The procedure was uneventful. Intraoperative oximetry reading varied between 82% and 86%. Congenital methemoglobinemia occurs due to deficiency of enzymes nicotinamide adenine dinucleotide (NADH) b5 reductase and nicotinamide adenine dinucleotide phosphate flavin reductase.[123] Individuals with G6PD deficiency are more susceptible as this enzyme helps in inhibition of oxidation of haemoglobin and reduction of methemoglobin.[2] Haemoglobin M disease is another congenital cause, in which substitution of amino acid in heme moiety occurs, which results in oxidised haemoglobin. Acquired methemoglobinemia is more common and occurs on exposure to oxidising agents such as aniline dyes, nitrobenzene, nitrate, nitrite, benzocaine, prilocaine, dapsone, pyridium, nitric oxide, nitrous oxide and naphthalene due to overwhelming of capacity of reducing enzymes.[2] Children are more susceptible due to the presence of foetal haemoglobin, which oxidises more easily, low levels of NADH reductase, higher gastric pH, which promotes growth of nitrate reducing organisms causing conversion of dietary nitrates to nitrite.[234] Clinically, this disorder is differentiated from cardiopulmonary diseases by lack of significant pulmonary and cardiac finding on history, examination and investigations and cyanosis, which fails to respond to supplemental oxygen. ABG reveals the classical ‘saturation gap’, with normal paO2 despite cyanosis.[2] Regardless of aetiology, symptoms appear depending on methemoglobin level in blood. Chocolate colour cyanosis presents at levels of 5-15% and appears early in anaemic patients. Nevertheless, at least 5 g of reduced haemoglobin must be present for cyanosis to be appreciated. At 30-40%, weakness, headache, dyspnoea, tachycardia and dizziness occurs. Patient will be lethargic, stuporous, confused and comatose at concentration of 55-60%. At 70% circulatory collapse occurs.[12] Intraoperative monitoring should ideally include co-oximetry, which detects the presence as well as quantifies methemoglobin level.[5] Pre-operative treatment with vitamin C helps in non-enzymatic reduction of methemoglobin. Injection methylene blue is the antidote and the dose is 1-2 mg/kg IV over 3-5 min.[23] It acts by increasing the level of NADH methemoglobin reductase, which helps in conversion of ferric ion to ferrous ion. Exchange transfusion and haemodialysis is indicated in severe cases. In acquired methemoglobinemia offending drug should be withdrawn and care should be taken to avoid further exposure to offending agent. Anaesthetic drugs, which induce methemoglobinemia are local anaesthetics, the “caines” (prilocaine, benzocaine, lidocaine, eutectic mixture of local anaesthetics cream), nitrous oxide, metoclopramide, nitroglycerine, sodium nitroprusside and should be avoided.[12] Predisposing factors for methemoglobinemia include age (infants under 6 months), the status of the area being sprayed or injected (inflamed areas and broken skin absorb more of the drug), concomitant use of other drugs which also have been implicated in causing methemoglobinemia and the genetic make-up of the patient (due to altered haemoglobin, G6PD deficiency or methemoglobin reductase enzyme deficiency). Use of local anaesthetic agents in lower doses may be an approach to be followed in this group of patients.[6] Heightened awareness of proper dosing and the risk of methemoglobinemia is particularly important for clinicians involved in endoscopy, intubation, bronchoscopy or similar invasive procedures using topical anaesthetic-containing sprays. Knowledge of the condition, appropriate monitoring and avoidance of the precipitating factors and availability of antidote are key factors in managing a case of methemoglobinemia.

A wide difference in susceptibility to nitrite between Eurasian perch (Perca fluviatilis L.) and largemouth bass (Micropterus salmoides Lac.)

Influence of nitrite on two fish species, Eurasian perch (Perca fluviatilis L.) and largemouth bass (Micropterus salmoides Lacepede), was assessed in two acute toxicity tests. In the first one, lethal concentrations (48hLC50) of nitrite were estimated at 11 mg l−1 NO2 − for perch and 882 mg l−1 NO2 − for bass. In the second test, fishes were exposed for 48 h to concentrations representing ¼ and ½ value of 48hLC50 for each species. This test showed that the higher nitrite concentration in the water the higher methaemoglobin content in the blood, and nitrite levels in the blood plasma were observed in both species. On the other hand, leucocyte count showed opposite trend. Activity of NADH-methaemoglobin reductase was markedly lower in largemouth bass compared to Eurasian perch and was stimulated by nitrite exposure in neither of the species.

Methaemoglobin Content and NADH-methaemoglobin Reductase Activity of Three Human Erythrocyte Genotypes

Background: To study methaemoglobin content and NADH-methaemoglobin reductase activity of three human erythrocyte genotypes (HbAA, HbAS and HbSS). Materials and Methods: Studies to ascertain methaemoglobin concentration and level of NADH-methaemoglobin reductase activity of three human erythrocyte genotypes (HbAA, HbAS and HbSS) were carried out in forty-three (43) healthy male participants of confirmed erythrocyte genotypes: HbAA (N=15), HbAS (N=15) and HbSS (N=13). Spectrophotometric method was used for determination of the two erythrocyte parameters. Results: Whereas methaemoglobin concentrations in the three erythrocyte genotypes was in the order: HbAS 0.05) in methaemoglobin concentration and NADH-methaemoglobin reductase activity between HbAA and HbAS erythrocytes. Conclusion: The results showed a relationship between erythrocyte NADH-methaemoglobin reductase activity and methaemoglobin concentration.

Ascaris suum NADH-methemo(myo)globin reductase systems recovering differential functions of hemoglobin and myoglobin, adapting to environmental hypoxia

We reported previously that Ascaris suum cytochrome b 5, specifically expressed in this nematode at the adult stage and dually localized in extracellular perienteric fluid and hypodermis, is involved in both perienteric NADH-methemoglobin and cytosolic NADH-metmyoglobin reduction, where cytochrome b 5 functions as an electron carrier between NADH-mediated cytochrome b 5 reductase and substrates, methemo(myo)globins to reduce the nonfunctional globins back to functional ferrous hemo(myo)globins. To further characterize NADH-methemo(myo)globin reductase systems, the midpoint potentials of A. suum perienteric hemoglobin and body wall myoglobin, as well as the affinities of Ascaris methemoglobin and metmyoglobin toward cytochrome b 5, were evaluated using potentiometric titration and surface plasmon resonance techniques, respectively. Midpoint potentials of + 7.2 mV and + 19.5 mV were obtained for Ascaris perienteric hemoglobin and body wall myoglobin, respectively. The affinities of Ascaris perienteric methemoglobin and body wall metmyoglobin toward the nematode cytochrome b 5 were comparable to that for mammalian hemoglobin and cytochrome b 5; association constants were 0.585 × 10 3 M − 1 and 2.32 × 10 3 M − 1 , respectively, with rapid equilibration kinetics. These observations highlight the physiological importance of A. suum perienteric NADH-methemoglobin and cytosolic metmyoglobin reductase systems. Differential roles of A. suum perienteric hemoglobin and body wall myoglobin are also discussed from the viewpoint of oxygen homeostasis under hypoxic conditions.

Antioxidant Status of Red Blood Cells Is a Modifying Factor of Clinical Manifestation of Unstable Hemoglobin Variant Hana [α2β2 63 (E7) His-Asn)

Hemoglobin Hana [α2β2 63 (E7) His-Asn)] was described in a Moravian family from the Czech Republic as an unstable hemoglobin variant with mild Heinz body hemolytic anemia, elevated levels of methemoglobin (12–13%), and variable reticulocytosis (1.8–11%) in the proband and her sister.1 Surprisingly, their mother is asymptomatic, although she expresses the same aberrant variant of the β-globin gene. We did not detect any differences in the globin gene expression (based on wild-type and mutant globin mRNA from reticulocytes and βA and βHana chains measured by HPLC).1 Iron metabolism indices were within normal range in all family members. G6PD status in both parents was normal. To test whether the erythrocyte antioxidant capacity affects severity of this unstable hemoglobinopathy, key antioxidant parameters, including content of reduced (GSH) and oxidized glutathione (GSSG), activity of superoxide dismutase (SOD), catalase (CAT), and NADH-methemoglobin reductase (MetHb RED) were examined. Our results revealed that erythrocytes of both children exhibited lower GSH content, the ratio of GSSG/GSH was increased 6 to 7 times, and their catalase activity was decreased to 71–77% of the average control activity (Table 1). The increased ratio of GSSG/GSH could be related to the accumulation of peroxides in erythrocytes of the children and reflects lower ability of the enzyme antioxidant system to eliminate free oxygen radicals. In contrast, erythrocytes of their mother exhibited normal values of all these measured parameters except the activity of SOD, which was increased by 32% above normal level (Table 1). Activities of some antioxidant enzymes are altered in cigarette smokers2 and the mother is a heavy smoker (with elevated CO-Hb to 5.5%). We hypothesize she might benefit from higher SOD activity caused by activation of “oxidative stress genes” and that could, in turn, enhance the ability of her erythrocytes to deal with superoxide and hydrogen peroxide accumulation which results in more efficient prevention of hemoglobin oxidation. In the children, the decrease in hydrogen peroxide catabolizing capacity due to lower erythrocyte catalase activity strongly associates with a more severe clinical manifestation of this unstable hemoglobinopathy. Our findings suggest that alterations (either inherent or caused by extrinsic factors) in the levels of red blood cell antioxidant defense enzymes represent modifying factors affecting the clinical manifestation of Heinz body hemolytic anemia associated with an unstable hemoglobin variant.Table 1:Levels of erythrocyte antioxidant systems in the family membersUnaffected MotherDaughter 1 with HADaughter 2 with HANormal controls (n = 10)RangeGSHa(μmol/g Hb)3.72.92.73.9±0.63.3–4.5GSSG/GSHb0.351.91.50.27±0.20.12–0.42MetHb RED (U/g Hb)15151416±2.813–19SOD (U/g Hb)2707232519332052±3111741–2363CAT (U/g Hb)282227245320±57263–377a - spectrophotometry, b - HPLC-MS

Inactivation of Human White Blood Cells in Red Blood Cell Products Using Mirasol® Pathogen Reduction Technology (PRT).

The transfusion of blood products can result in transmission of pathogens and immunological consequences such as transfusion associated graft-versus-host disease and alloimmunization. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids that results in inactivation of a wide range of pathogens as well as inhibition of the immunological responses mediated by the WBC present in platelet concentrates. Initial studies investigated the effects of Mirasol PRT treatment of RBC concentrates on WBC function. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation from control or test non-leukoreduced RBC units exposed to riboflavin and varying doses of light. These PBMNC were tested for their ability to be activated in response to PMA, to proliferate in response to PHA or allogeneic stimulator cells, and to stimulate proliferative responses of allogeneic responder cells. While lower light doses were sufficient to inhibit activation and proliferation, higher energies (30J/ml RBC) were required for inhibition of antigen presentation. These Mirasol treatment conditions did not induce crossmatch incompatibility, methemoglobin (MetHb) levels stayed within an acceptable range (2.6% + 1.5) and hemolysis was below 1% during storage. Interestingly, MetHb levels decreased to background levels during storage at 4°C, suggesting that the activity of the enzyme NADH methemoglobin reductase was not compromised by the exposure to riboflavin and light. These treatment conditions also reduced levels of Gram positive and negative bacteria to the limits of detection and reduced infectivity of both enveloped and non-eveloped viruses by > 4 logs on average in packed, washed RBC units. In summary, Mirasol PRT is able to functionally inactivate WBCs and pathogens in washed RBC products without adversely affecting the quality of the RBCs. This novel pathogen reduction therapy for RBC products complements the Mirasol PRT treatment for platelets and offers inactivation of pathogens in cellular blood components using a single technology.

Congenital methemoglobinemia caused by Hb‐MRatnagiri (β‐63CAT→TAT, His→Tyr) in an Indian family

Hb-M is a very rare hemoglobinopathy in the Indian subcontinent. We report a family with Hb-M with lifelong cyanosis from the Ratnagiri district in western India. The propositus was a 11-year-old female child with a history of increasing cyanosis exacerbated by fever and weakness. Similar complaints were also noted in her mother and five maternal family members. There was no history of cardiac illness or exposure to drugs and chemicals. The methemoglobin level was 39.3% in the propositus and 21.1% in her mother with normal NADH-methemoglobin reductase activity. Abnormal absorption peaks by spectroscopic analysis, presence of hemoglobin instability, and a slow-moving band on starch gel electrophoresis supported the presence of Hb-M. Automated DNA sequence analysis of the beta globin gene showed a C-->T substitution at codon 63. This leads to a substitution of histidine (CAT) by tyrosine (TAT) at the beta 63 (E7) position, similar to Hb-M Saskatoon. We have named this variant as Hb-M(Ratnagiri).

Investigation of methaemoglobin reduction by extracellular NADH in mammalian erythrocytes

The effect of extracellular NADH on the rate of reduction of nitrite-induced methaemoglobin in erythrocytes from man, cattle, dog, horse, grey kangaroo, pig and sheep was investigated. Extracellular NADH was found to enhance the rate of methaemoglobin reduction in man, dog, pig and kangaroo erythrocytes, but had essentially no effect on the rate of methaemoglobin reduction in erythrocytes from cattle, horse and sheep. In erythrocytes of those animals affected by extracellular NADH the rate of reduction of metHb in the presence of NADH was the same or greater than that observed in the presence of nutrients such as glucose and inosine. The combination of nutrient and NADH produced a more profound increase in the rate of methaemoglobin reduction. The rate of methaemoglobin reduction in all cases was significantly less than that observed with methylene blue, the standard treatment of methaemoglobinaemia. Extracellular NADH was found to indirectly increase the intracellular NADH concentration through displacement of the pseudo-equilibrium of the intracellular LDH reaction and relied upon the presence of sufficient LDH activity released into the extracellular medium through haemolysis. The lack of response of cattle, horse and sheep RBCs to extracellular NADH was found to derive mainly from their low extracellular LDH activity, but also correlated with their lower NADH-methaemoglobin reductase activity compared to the other species.

Hematological responses of the Neotropical teleost matrinxã ( Brycon cephalus) to environmental nitrite

Environmental increase in nitrite impairs the function of several aquatic species, including fishes. Nitrite reacts with hemoglobin yielding the non-functional methemoglobin (metHb), and many physiological disturbances can arise. The physiological mechanisms to cope with nitrite are still unclear in fish. Hematological parameters, the role of NADH–methemoglobin reductase system and the electrolytic balance were studied in the freshwater teleost Brycon cephalus (matrinxã) exposed to 0.2, 0.4 and 0.6 mg/L of nitrite N–NO 2 for 24 and 96 h. Hematocrit, total hemoglobin and the red blood cell (RBC) number decreased. Methemoglobin content increased from 1% to 69% for 24 h of exposure and drastically from 5–6% to 90% for 96 h. The activity of NADH–methemoglobin reductase system displayed a tendency of increase in response to nitrite concentration or time of exposure. In the plasma, nitrite was accumulated to values 30-fold higher than the environmental concentration. The plasma K + concentration increased only in fish exposed to NO 2 for 24 h. No changes in plasma protein and Na + were observed during nitrite exposure but Cl-presented a punctual increase at 0.2 mg/L N–NO 2–96 h. The hematological data suggest that nitrite caused functional and hemolytic anemia. Furthermore, the electrolytic balance was relatively undisturbed, and the nitrite clearance in matrinxã is likely depending on other factors than NADH–methemoglobin reductase system.

Activity of membrane-bound NADH-methemoglobin reductase and physical state of lipids in erythrocyte membranes

The activity of membrane-bound NADH-methemoglobin reductase in erythrocytes and the physical state of lipids in erythrocyte membranes under oxidative stress in cells were studied. A decrease of the activity of membrane-bound NADH-methemoglobin reductase and a change of physical state of the lipid bilayer of membranes under oxidative stress were found in erythrocytes in vivo and in vitro.

Methemoglobin Interferes with the HemoCue B-Glucose Analyzer

During the investigation of a neonate with NADH-methemoglobin (NADH-MetHb) reductase deficiency (baseline MetHb, 28%), we noticed disparate readings between the ward-based, whole-blood HemoCue B-Glucose Analyzer (HemoCue AB) and the main laboratory plasma glucose method (glucose oxidase) performed on a Synchron CX7 Delta analyzer (Beckman Coulter). The HemoCue results suggested profound hypoglycemia, which was out of keeping with the clinical status of the patient, whereas the Synchron CX7 Delta results were consistently within the pediatric reference interval. These findings were contrary to the previously documented overestimation of the HemoCue analyzer at low glucose concentrations (1)(2)(3). Because the HemoCue analyzer (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) uses a mutarose–glucose dehydrogenase enzyme system to produce a colored formazan, which is then quantified photometrically, the possibility of spectral interference by MetHb with the HemoCue analyzer was raised and investigated …

Use of methylene blue to treat methemoglobinemia in infancy.

AN ABNORMALLY INCREASED concentration of methemoglobin in the blood (methemoglobinemia) occurs whenever there is an imbalance between oxidation and reduction of heme iron. Methemoglobin is a form of hemoglobin (the oxygen-carrying pigment of the red blood cells) in which heme iron has been oxidized to the ferric state, making it unable to transport oxygen (Table 1). Normally, methemoglobin is continuously formed at a slow rate by the oxidation of heme iron to the ferric state and is then enzymatically reduced back to hemoglobin. The enzyme NADH-methemoglobin reductase accounts for nearly all methemoglobin reduction. Other mechanisms for methemoglobin reduction include antioxidants such as vitamin C and glutathione. Methemoglobinemia occurs when more than 1 percent of hemoglobin is oxidized to methemoglobin; cyanosis occurs with methemoglobin levels of approximately 1.5 gm/dl.1 Table 2 shows the normal methemoglobin concentrations for infants of various ages.

Erythrocyte enzyme activities in cord blood of extremely low-birth-weight infants.

To investigate the features of erythrocyte metabolism in extremely immature infants, we assayed 21 enzyme activities and glutathione level in cord erythrocytes from 28 extremely low-birth-weight infants (ELBWI; defined as birth weight <1,000 g). The results were compared with those from normal adults and non-neonatal reticulocyte-rich controls. Statistical analysis revealed that activities of six enzymes (glucosephosphate isomerase, phosphoglycerate kinase, monophosphoglycerate mutase, enolase, glucose-6-phosphate dehydrogenase (G6PD), and glutathione reductase) were significantly higher, and those of eight other enzymes (phosphofructokinase, 6-phosphogluconate dehydrogenase (6PGD), glutathione peroxidase, adenylate kinase, adenosine deaminase, acetylcholinesterase, NADH methemoglobin reductase, and catalase) were lower in ELBWI taking their marked reticulocytosis into consideration. The 6PGD/G6PD ratio, which is consistently unchanged under various physiological and pathological conditions, was markedly reduced in ELBWI. Our results support the previous reports that neonatal erythrocytes have a unique metabolic pattern which is different from that of adult erythrocytes, and also suggest that the 6PGD/G6PD ratio might be an index for the developmental immaturity of fetal erythrocytes. This is the first report describing the pattern of erythrocyte enzyme activities in ELBWI.